Project Summary Cardiovascular diseases represent the number one cause of morbidity and mortality worldwide, affecting a broad spectrum of ages from babies that are born with congenital heart defects (CHDs) to adults that suffer acute myocardial infarctions and/or develop congestive heart failure over time. My research program is motivated by the simple assumption that we can use the zebrafish model organism to understand on a molecular and cellular level how the cardiovascular system is established during development and how it regenerates during adulthood. Here, I plan to leverage the longer-term support and increased scientific flexibility afforded by the NHLBI R35 Emerging Investigator Award to continue and expand my laboratory?s two main research focuses in great vessel morphogenesis and heart regeneration to address significant challenges in each field. Specifically, I will use the genetic and regenerative attributes of the zebrafish system: (1) to model human CHDs that disrupt great vessel establishment and, (2) to uncover critical barriers to mammalian heart regeneration. In regards to the former, we have made paradigm-shifting observations concerning the cellular etiology of the cardiovascular phenotypes present in DiGeorge Syndrome patients that are caused by hemizygous microdeletions on chromosome 22q11.2, a region that harbors the TBX1 gene. We plan to delve deeper into these cellular mechanisms and break new ground by identifying biologically relevant targets of the Tbx1 transcriptional complex using a proprietary knock-in zebrafish strain. As ~20% of individuals carrying the 22q11.2 deletion lack any discernable pathology, we anticipate that the genes we discover as Tbx1 transcriptional targets will represent novel candidates that profoundly influence the severity of DGS cardiovascular defects. We also plan to create new CHD models in an effort to link previously identified genetic variants that segregate with great vessel CHDs in newborns to the pathology and to uncover the cellular and molecular basis of disease. The outcomes of the proposed studies will contribute significantly to our fund of knowledge and likely influence genetic counseling, pre-natal diagnosis, and possibly pre-natal repair. Additionally, we have uncovered novel determinants of myocardial proliferation in regenerating zebrafish hearts that likely contribute to the regenerative failures observed in mammalian hearts, including humans. Specifically, we plan to further explore the required role of Notch signaling in zebrafish heart regeneration, understand how alterations in the epigenetic landscape and in chromatin accessibility influence cardiomyocyte proliferation, and determine whether myocardial ploidy affects regenerative capacity. The outcomes of the proposed studies will directly guide future approaches to coax mammalian hearts towards regeneration instead of scarring and identify practical inroads for regenerative medicine. !